Cytoplasmic calcium ([Ca2+]i) provided through voltage-dependent Ca2+ channels (VDCC) plays an important role in adrenocorticotropin (ACTH)-induced steroidogenesis in adrenocortical cells. To identify alternative mechanisms for [Ca2+]i supply, we investigated the 2-aminoethoxydiphenyl borate (2APB)-sensitive pathway as one of the possible signaling pathways involved in [Ca2+]i supply for ACTH-induced steroidogenesis. In monolayers of cultured rat adrenal fasciculate and reticularis cells, ACTH at 10-11 M stimulated corticosterone synthesis without increasing intracellular cAMP, and corticosterone synthesis was decreased by 10 μ M 2APB by 51.8% (6.71 ± 0.97 vs. 3.23 ± 0.05 ng/mL/4hours; p<0.05). Furthermore, 2APB significantly decreased the 10-11 M ACTH-stimulated [Ca2+]i. ACTH increased the intracellular inositol-1,4,5-trisphosphate (IP3) content with a peak at 10-13 M ACTH, which illustrates the possibility that ACTH activates IP3/diacylglycerol- dependent protein kinase C signal transduction. However, the difference in ACTH concentrations between that responsible for the IP3 increase and steroidogenesis without elevated cAMP, suggest a hypothesis that IP3 is not required for steroidogenesis, but does involve an unknown messenger, which stimulates the release of Ca2+ from the ER or the subsequent store-operated Ca2+ entry (SOCE). The pregnenolone concentration in the culture medium was increased by ACTH, which was significantly suppressed by 2APB, showing that the 2APB-sensitive Ca2+ supply affects cholesterol transport into the mitochondrial membrane via steroidogenic acute regulatory protein. Therefore, the SOCE may contribute to ACTH-induced steroidogenesis in the mitochondrial region. In conclusion, the [Ca2+]i used for steroidogenesis may be derived from a 2APBsensitive pathway and via VDCCs, particularly at physiological concentrations of ACTH. We suggest that ACTH receptors activate steroidogenesis via inositol triphosphate, or an unknown downstream messenger, which could be inhibited by 2APB.